RRM
2011 Rustbelt RNA Meeting
RRM
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Poster abstracts
Abstract:
The appearance in erythroid cells of stable 5’-truncated products of nonsense-containing β-globin mRNA (1) was the first evidence for endonuclease cleavage in NMD. These decay products are independent of PTC position and their appearance is linked to the PMR1 endonuclease(3). They were originally detected using S1 protection; however, this assay is time-consuming, insensitive, difficult to quantify and difficult to apply to large numbers of samples. To monitor changes in full-length and decay products following overexpression and knockdown of proteins in the decay process we developed a highly sensitive assay that combines 5’-linker ligation and 5’ RACE termed MBRACE (3). The first steps involve cap hydrolysis with TAP or Dcp2, and selection of poly(A) RNA. The 5’-monophosphate ends are then ligated to an RNA linker and the product is next converted into an oligo(dT)-primed cDNA. Finally, the modified MBRACE qPCR is performed using a forward primer matching the ligated RNA oligo, a reverse primer downstream within β-globin mRNA and two dual-labeled molecular probes that are specific for the junction between the linker sequence and either the uncapped 5’-end of the full-length mRNA or the 5’ end of one of the identified decay intermediates. Data are normalized to β-actin as determined by SYBR Green qPCR. Data will be presented showing the applicability of this to studying the impact of altering the location of the PTC on the degree of endonuclease cleavage and of knocking down Upf1 and SMG6 on this endonuclease-mediated process.
References:
1. Lim S-K et al. (1992) Molecular and Cellular Biology. 12:3; 1149-1161.
2. Bremer et al. (2003) RNA. 9: 1157-1167.
3. Lasham A et al. (2009) Nucleic Acids Research. 38:3 e19
Keywords: nonsense-mediated mRNA decay, endonucleolytic decay
